The present invention relates to novel indane and indene derivatives, pharmaceutical compositions containing these compounds and their use as endothelin receptor antagonists.
Endothelin (ET) is a highly potent vasoconstrictor peptide synthesized and released by the vascular endothelium. Endothelin exists as three isoforms, ET-1, ET-2 and ET-3. [Unless otherwise stated xe2x80x9cendothelinxe2x80x9d shall mean any or all of the isoforms of endothelin]. Endothelin has profound effects on the cardiovascular system, and in particular, the coronary, renal and cerebral circulation. Elevated or abnormal release of endothelin is associated with smooth muscle contraction which is involved in the pathogenesis of cardiovascular, cerebrovascular, respiratory and renal pathophysiology. Elevated levels of endothelin have been reported in plasma from patients with essential hypertension, acute myocardial infarction, subarachnoid hemorrhage, atherosclerosis, and patients with uraemia undergoing dialysis.
In vivo, endothelin has pronounced effects on blood pressure and cardiac output. An intravenous bolus injection of ET (0.1 to 3 nmol/kg) in rats causes a transient, dose-related depressor response (lasting 0.5 to 2 minutes) followed by a sustained, dose-dependent rise in arterial blood pressure which can remain elevated for 2 to 3 hours following dosing. Doses above 3 nmol/kg in a rat often prove fatal.
Endothelin appears to produce a preferential effect in the renal vascular bed. It produces a marked, long-lasting decrease in renal blood flow, accompanied by a significant decrease in GFR, urine volume, urinary sodium and potassium excretion. Endothelin produces a sustained antinatriuretic effect, despite significant elevations in atrial natriuretic peptide. Endothelin also stimulates plasma renin activity. These findings suggest that ET is involved in the regulation of renal function and is involved in a variety of renal disorders including acute renal failure, cyclosporine nephrotoxicity, radio contrast induced renal failure and chronic renal failure.
Studies have shown that in vivo, the cerebral vasculature is highly sensitive to both the vasodilator and vasoconstrictor effects of endothelin. Therefore, ET may be an important mediator of cerebral vasospasm, a frequent and often fatal consequence of subarachnoid hemorrhage.
ET also exhibits direct central nervous system effects such as severe apnea and ischemic lesions which suggests that ET may contribute to the development of cerebral infarcts and neuronal death.
ET has also been implicated in myocardial ischernia (Nichols etal. Br. J. Pharm. 99: 597-601, 1989 and Clozel and Clozel, Circ. Res., 65: 1193-1200, 1989) coronary vasospasm (Fukuda et al., Eur. J. Pharm. 165: 301-304, 1989 and Lxc3xcscher, Circ. 83: 701, 1991) heart failure, proliferation of vascular smooth muscle cells, (Takagi, Biochem and Biophys. Res. Commun.; 168: 537-543, 1990, Bobek et al., Am. J. Physiol, 258:408-C 415, 1990) and atherosclerosis, (Nakaki et al., Biochem. and Biophys. Res. Commun. 158: 880-881, 1989, and Lerman et al., New Eng. J. of Med. 325: 997-1001, 1991). Increased levels of endothelin have been shown after coronary balloon angioplasty (Kadel et al., No. 2491 Circ. 82: 627, 1990).
Further, endothelin has been found to be a potent constrictor of isolated mammalian airway tissue including human bronchus (Uchida et al., Eur J. of Pharm. 154: 227-228 1988, LaGente, Clin. Exp. Allergy 20: 343-348, 1990; and Springall et al., Lancet, 337: 697-701, 1991). Endothelin may play a role in the pathogenesis of interstitial pulmonary fibrosis and associated pulmonary hypertension, Glard et al., Third International Conference on Endothelin, 1993, p. 34 and ARDS (Adult Respiratory Distress Syndrome), Sanai et al., Supra, p. 112.
Endothelin has been associated with the induction of hemorrhagic and necrotic damage in the gastric mucosa (Whittle et al., Br. J. Pharm. 95: 1011-1013, 1988); Raynaud""s phenomenon, Cinniniello et al., Lancet 337: 114-115, 1991); Crohn""s Disease and ulcerative colitis, Munch et al., Lancet, Vol. 339, p. 381; Migraine (Edmeads, Headache, February 1991 p 127); Sepsis (Weitzberg et al., Circ. Shock 33: 222-227, 1991; Pittet et al., Ann. Surg. 213: 262-264, 1991), Cyclosporin-induced renal failure or hypertension (Eur. J. Pharmacol., 180: 191-192, 1990, Kidney Int, 37: 1487-1491, 1990) and endotoxin shock and other endotoxin induced diseases (Biochem. Biophvs. Res. Commun., 161: 1220-1227, 1989, Acta Physiol. Scand. 137: 317-318, 1989) and inflammatory skin diseases, (Clin Res. 41:451 and 484, 1993) and macular degeneration.
Endothelin has also been implicated in preclampsia of pregnancy. Clark et al., Am. J. Obstet. Gynecol, March 1992, p. 962-968; Kamor et al., N. Eng. J. of Med., Nov. 22, 1990, p. 1486-1487; Dekker et al., Eur J. Ob. and Gyn. and Rep. Bio. 40 (1991) 215-220; Schiff et al., Am. J. Obstet. Gynecol. February 1992, p. 624-628; diabetes mellitus, Takahashi et al., Diabetologia (1990) 33:306-310; and acute vascular rejection following kidney transplant, Watschinger et al., Transplantation Vol. 52, No. 4, pp. 743-746.
Endothelin stimulates both bone resorption and anabolism and may have a role in the coupling of bone remodeling. Tatrai et al. Endocrinology, Vol. 131, p. 603-607.
Endothelin has been reported to stimulate the transport of sperm in the uterine cavity, Casey et al., J. Clin. Endo and Metabolism, Vol. 74, No. 1, p. 223-225, therefore endothelin antagonists may be useful as male contraceptives. Endothelin modulates the ovarian/menstrual cycle, Kenegsberg, J. of Clin. Endo. and Met., Vol. 74, No. 1, p. 12, and may also play a role in the regulation of penile vascular tone in man, Lau et al., Asia Pacific J. of Pharm., 1991, 6:287-292 and Tejada et al., J. Amer. Physio. Soc. 1991, H1078-1085. Endothelin also mediates a potent contraction of human prostatic smooth muscle, Langenstroer et al., J. Urology, Vol. 149, p. 495-499.
Thus, endothelin receptor antagonists would offer a unique approach toward the pharmacotherapy of hypertension, renal failure, ischemia induced renal failure, sepsis-endotoxin induced renal failure, prophylaxis and/or treatment of radio-contrast induced renal failure, acute and chronic cyclosporin induced renal failure, cerebrovascular disease, myocardial ischemia, angina, congestive heart failure, asthma, atherosclerosis, macular degeneration, Raynaud""s phenomenon, ulcers, sepsis, migraine, glaucoma, endotoxin shock, endotoxin induced multiple organ failure or disseminated intravascular coagulation, cyclosporin-induced renal failure and as an adjunct in angioplasty for prevention or treatment of restenosis, diabetes, preclampsia of pregnancy, bone remodeling, kidney transplant, male contraceptives, infertility and priaprism and benign prostatic hypertrophy.
This invention comprises indane and indene derivatives represented by Formula (I) and pharmaceutical compositions containing these compounds, and their use as endothelin receptor antagonists which are useful in the treatment of a variety of cardiovascular and renal diseases including but not limited to: hypertension, acute and chronic renal failure, cyclosporine induced nephrotoxicity, stroke, cerebrovascular vasospasm, myocardial ischemia, angina, heart failure, atherosclerosis, and as an adjunct in angioplasty for prevention of restenosis and benign prostatic hypertrophy.
This invention further constitutes a method for antagonizing endothelin receptors in an animal, including humans, which comprises administering to an animal in need thereof an effective amount of a compound of Formula (I).
The compounds of this invention are represented by structural Formula (I): 
wherein:
R1 is xe2x80x94X(CH2)nAr or xe2x80x94X(CH2)nR8 or 
R2 is hydrogen, Ar, C1-4alkyl or (c);
P1 is xe2x80x94X(CH2)nR8;
P2 is xe2x80x94X(CH2)nR8, or xe2x80x94Xxe2x80x94R9xe2x80x94Y;
R3 and R5 are independently hydrogen, R11, OH, C1-8alkoxy, S(O)qR11, N(R6)2, Br, F, I, Cl, CF3, NHCOR6, R13CO2R7, xe2x80x94Xxe2x80x94R9xe2x80x94Y, or xe2x80x94X(CH2)nR8 wherein each methylene group within xe2x80x94X(CH2)nR8 may be unsubstituted or substituted by one or two xe2x80x94(CH2)nAr groups;
R4 is hydrogen, R11, OH, C1-5alkoxy, S(O)qR11, N(R6)2, Br, F, I, Cl or NHCOR6 wherein the C1-5alkoxy may be unsubstituted or substituted by OH, methoxy or halogen;
R6 is independently hydrogen or C1-4alkyl;
R7 is independently hydrogen, C1-10alkyl, C2-10alkenyl or C2-8alkynyl, all of which may be unsubstituted or substituted by one or more OH, N(R6)2, CO2R12, halogen or XC1- 5alkyl; or R7 is (CH2) nAr;
R8 is hydrogen, R11, CO2R7, CO2C(R11)2 O(CO)XR7, PO3(R7)2, SO2NR7R11, NR7SO2R11, CONR7SO2R11, SO3R7, SO2R7, P(O)(OR7)R7, CN, xe2x80x94CO2(CH2)mC(O)N(R6)2, C(R11)2N(R7)2, C(O)N(R6)2, tetrazole or OR6;
R9 is a bond, C1-10alkylene, C1-10alkenylene, C1-10alkylidene, C1-10alkynylene, all of which may be linear or branched, or phenylene, all of which may be unsubstituted or substituted by one or more OH, N(R6)2, COOH or halogen;
R10 is R3or R4;
R11 is hydrogen, Ar, C1-8alkyl, C2-8alkenyl, C2-8alkynyl, all of which may be unsubstituted or substituted by one or more OH, CH2OH, N(R6)2 or halogen;
R12 is hydrogen, C1-6alkyl, C2-6alkenyl or C2-7alkynyl;
R13 is divalent Ar, C1-10alkylene, C1-10alkylidene, C2-10alkenylene, C2-10alkynylene, all of which may be unsubstituted or substituted by one or more OH, CH2OH, N(R6)2 or halogen;
X is (CH2)n, O, NR6 or S(O)q;
Y is CH3 or X(CH2)nAr;
Ar is: 
naphthyl, indolyl, pyridyl, thienyl, oxazolidinyl, oxazolyl, thiazolyl, isothiazolyl, pyrazolyl, triazolyl, tetrazolyl, imidazolyl, imidazolidinyl, thiazolidinyl, isoxazolyl, oxadiazolyl, thiadiazolyl, morpholinyl, piperidinyl, piperazinyl, pyrrolyl, or pyrimidyl; all of which may be unsubstituted or substituted by one or more R3 or R4 groups;
A is Cxe2x95x90O, or (C(R6)2)m ;
B is xe2x80x94CH2xe2x80x94or xe2x80x94Oxe2x80x94;
Z1 and Z2 are independently hydrogen, C1-8alkyl, C2-8alkenyl, C2-8alkynyl, OH, C1-8alkoxy, S(O) q C1-8alkyl, N(R6)2, Br, F, I, Cl, NHCOR6, xe2x80x94Xxe2x80x94R9xe2x80x94Y, xe2x80x94X(CH2)nR8, phenyl, benzyl or C3-6cycloalkyl wherein the C1-8alkyl, C2-8alkenyl or C2-8alkynyl may be optionally substituted by COOH, OH, CO(CH2)nCH3, CO(CH2)nCH2N(R6)2, or halogen; or Z1 and Z2 together may be xe2x80x94Oxe2x80x94Axe2x80x94Oxe2x80x94 on contiguous carbons;
Z3 is Z1 or xe2x80x94Xxe2x80x94R9xe2x80x94Y;
q is zero, one or two;
n is an integer from 0 to six;
m is 1, 2 or 3;and the dotted line indicates the optional presence of a double bond; or a pharmaceutically acceptable salt thereof; provided that
R2 is not hydrogen when X is S(O)q;
when the optional double bond is present there is only one R10 and there is no P1 and P2 is not NR6R9Y; and if Xxe2x80x94R2 is attached to the double bond, X is not NR6; and if R1 is attached directly to the double bond, R1 is not NR6AR;
when R3, R5, Z1, Z2, or Z3 is X(CH2)nR8 and n is not 0, X is oxygen or NR6 when R8 is OR6 or CO2H;
when R8 is CO2C(R11)2O(CO)XR7, X is not S(O)q;
the compound of Formula I is not (1RS)-1,3-diphenylindene-2-carboxylic acid; (cis,cis)-(1RS,3SR)-1,3-diphenylindane-2-carboxylic acid; (1RS)-3-[3-Methyl-1-phenyl-(1H)-ind-2-en-1-yl] propionic acid; or (1RS)-2-[1,3-diphenyl-(1H)-ind-2-en-2-yl]ethanoic acid; 1,3-diphenyl-1-ethoxyindene-2-carboxylic acid; 1,2,3-triphenylindene; 1,3 diphenylindene; 1-(2,3-dimethyl-2-buten-yl)-1,3-diphenylindene; 1,3-diphenyl-2-methylindene; 1,3-diphenyl-2-methylindane; 1,3-diphenylindane; 5,6-dimethoxy-1,3-dimethoxyindene; 1,3-bis(4,5-dimethoxy-2-hydroxyphenyl)-5,6-dimethoxyindane; 1,3-bis(3,4-dimethoxyphenyl)-5,6-dimethoxyindane; 1,3-diphenyl-2-methoxyidene, 1,3-diphenyl-2-ethoxyindene, 5-fluoro-2-methyl-indene-3-acetic acid, methyl 1,3-diphenylindene-2-carboxylate, ethyl 1,3-diphenylindene-2-carboxylate, or 2-cyano-1,3-diphenylindene.
Also included in the invention are pharmaceutically acceptable salt complexes. Preferred are the ethylene diamine, sodium, potassium, calcium and ethanolarnine salts.
The term alkylene is a divalent alkyl group in which the bonds are on two different carbon atoms; alkylidene is a divalent alkyl group in which the bonds are on the same carbon atom; alkenylene is a divalent alkene group in which the bonds may be on any carbon atom; alkynylene is a divalent alkynyl group in which the bonds may be on any carbon atom.
All alkyl, alkenyl, alkynyl, alkoxy, alkylene, alkylidene, alkenylene and alkynylene groups may be straight or branched. The term xe2x80x9chalogenxe2x80x9d is used to mean iodo, fluoro, chloro or bromo. Alkyl groups may be substituted by one or more halogens up to perhalocenation.
The compounds of the present invention may contain one or more asymmetric carbon atoms and may exist in racemic and optically active form. All of these compounds and diastereoisomers are contemplated to be within the scope of the present invention.
Preferred compounds are those wherein R1 is X(CH2)nAr, (Ar is (a) or (b)), dihydrobenzofuranyl, benzodioxanyl, cyclohexyl or C1-4alkyl; R2 is a moiety of formula (a) or (b), C1-4alkyl, indolyl or hydrogen; R3 and R5 are independently hydrogen, OH, C1-5alkoxy, halogen, xe2x80x94OC1-4alkyl phenyl, R13CO2R7, C1-4alkyl, N(R6)2, NH(CO)CH3, xe2x80x94X(CH2)nR8, xe2x80x94Xxe2x80x94R9xe2x80x94Y pyridyl, phenyl or S(O)pC1-5alkyl; R4 is hydrogen, OH, C1-5alkoxy, halogen, C1-4alkyl, N(R6)2, NH(CO)CH3 or S(O)pC1-5alkyl; Z1, Z2 and Z3 are independently XR9Y, benzyl, hydrogen, OH, C1-5alkoxy, xe2x80x94N(R6)2, S(O)qC1-8alkyl, NHCOR6, X(CH2)nR8 or halogen, or Z1 and Z2 together may be xe2x80x94Oxe2x80x94Axe2x80x94O on contiguous carbons; P1 and P2 are independently hydrogen, CO2H, C(R6)2CO2H or tetrazole; Ar is a moiety of formula (a) or (b), phenyl, or pyridyl; X is (CH2)n or oxygen.
More preferred are compounds wherein R3 is hydrogen, xe2x80x94X(CH2)nR8 or R13CO2R7; R4 and R5 are independently hydrogen, OH, C1-5alkoxy, SC1-5alkyl, substituted phenyl, F, Br, C1-3alkyl or NH2; Z1 and Z3 are hydrogen and Z2 is hydrogen, OH, C1-5alkoxy, halogen, X(CH2)nR8, NH2, benzyl, NH(CO)CH3, or Z1 and Z2 together may be Oxe2x80x94Axe2x80x94O on contiguous carbons and R1, R2, P1, P2, Ar and X are as above for preferred compounds.
Most preferred are compounds wherein R1 is (b) and R2 is (a) or (b); A is CH2, B is xe2x80x94Oxe2x80x94; there is no optional double bond; R1 and XR2 are trans to P1; Z2 is hydrogen, OH, C1-5alkoxy, or xe2x80x94OCH2CHxe2x95x90CH2, Z1 is hydrogen; R3 is XAr, hydrogen, X(CH2)nCOOH, X(CH2)nCONR7SO2R11, X[(CR6)2]nOR6 or CHxe2x95x90CHCO2H; R4 is hydrogen, substituted phenyl, pyridyl or pyrimidyl, or C1-2alkoxy; R5, R10 and P2 are hydrogen, and P1 is CO2H or C(R6)2CO2H.
Especially preferred are compounds wherein R1 is (b) and R2 is (a); A is CH2, B is xe2x80x94Oxe2x80x94; there is no optional double bond; R1 and XR2 are trans to P1; X is a bond; Z1 and Z3 are hydrogen; Z2 is hydrogen, OH or C1-5alkoxy; R3 is hydrogen, OAr (where Ar is (a), (b), pyridyl or pyrimidyl and A is CH2 and B is xe2x80x94Oxe2x80x94 and Ar may be substituted by CO2H), O(CH2)1-3CO2H, O(CH2)1-3CONHSO2R11, (CH2)0-4CO2H, (CH2)0-3CONH SO2R11, or O[(CR6)2]2-4 OH; R4 is hydrogen, C1-2alkoxy, or phenyl, pyridyl or pyrimridyl all of which may be substituted by R3 or C1-2alkoxy; R5, R10 and P2 are hydrogen; and P1 is CO2H or CH2CO2H.
Especially preferred compounds are the following:
(1RS ,2SR,3RS)-3-(2-Carboxymethoxy-4-methoxyphenyl)-1-(3,4-methylenedioxyphenyl)-5-(prop-1-yloxy)-indane-2-carboxylic acid;
(+)(1S ,2R,3S)-3-(2-Carboxymethoxy-4-methoxyphenyl)-1-(3,4-methylenedioxyphenyl)-5-(prop-1-yloxy)-indane-2-carboxylic acid;
(+)(1S ,2R,3S)-3-(2-Carboxymethoxy-4-methoxyphenyl)-1-(3,4-methylenedioxyphenyl)-5-(prop-1-yloxy)-indane-2-carboxylic acid disodium salt;
(1RS,2SR,3SR)-3-(2-Carboxymethoxy-4-methoxyphenyl)-1-(2-methoxy-4,5-methylenedioxyphenyl)-5-(prop-1-yloxy)-indane-2-carboxylic acid;
(1RS,2SR,3RS)-3-[2-(2-Carboxyeth-1-yloxy]-4-methoxyphenyl]-1-(3,4-methylenedioxyphenyl)-5-(prop-1-yloxy)-indane-2-carboxylic acid;
(1RS,2SR,3SR)-3-[2-[(E)-2-Carboxyethen-1-yl]-4-methoxyphenyl]-1-(3,4-methylenedioxyphenyl)-5-(prop-1-yloxy)indane-2-carboxylic acid;
(1RS,2SR,3SR)-3-[2-(2-Carboxyeth-1-yl)-4-methoxyphenyl]-1-(3,4-methylenedioxyphenyl)-5-(prop-1-yloxy)-indane-2-carboxylic acid;
(1RS,2SR,3RS)-3-[2-(3-Carboxyphenyl)-4-methoxyphenyl]-1-(3,4-methylenedioxyphenyl)-5-(prop-1-yloxy)indane-2-carboxylic acid;
(+)(1S,2R,3S)-3-[2-[(4-Carboxypyridin-3-yl)oxy]-4-methoxyphenyl]-1-(3,4-methylenedioxyphenyl)-5-(prop-1-yloxy)indane-2-carboxylate;
(+)(1S,2R,3S)-3-[2-(2-Hydroxyeth-1-yloxy)-4-methoxyphenyl]-1-(3,4-methylenedioxyphenyl)-5-(prop-1-yloxy)indane-2-carboxylate;
(1RS,2S R,3RS)-3-[2-(2-Hydroxyeth-1-yloxy)-4-methoxyphenyl]-1-(3,4-methylenedioxyphenyl)-5-(prop-1-yloxy)indane-2-carboxylate;
(+)(1S,2R,3S)-3-[2-(2-Hydroxyeth-1-yloxy)-4-methoxyphenyl]-1-(3,4-methylenedioxyphenyl)-5-(prop-1-yloxy)indane-2-carboxylate hemiethylenediamine salt;
(1RS,2SR,3RS)-3-(2-carboxymethoxy-4-methoxyphenyl)-1-(3,4-methylenedioxyphenyl)-5-(prop-1-yloxy)indane-2-yl-acetic acid.
(1RS,2SR,3RS)-3-[2-(2-Hydroxyeth-1-yloxy)-4-methoxyphenyl]-1-(3,4-methylenedioxyphenyl)-5-(prop-1-yloxy)indan-2-yl acetic acid
The present invention provides compounds of Formula (I) above 
which can be prepared by a process which comprises:
a) reacting a compound of Formula (2) wherein X is C1-5alkyl 
with a substituted benzaldehyde or aldehyde of Formula (3).
Dxe2x80x94CHOxe2x80x83xe2x80x83(3)
wherein D is Ar or (c) as defined in Formula I, in a suitable solvent such as benzene with a catalyst such as piperidinium acetate at reflux to provide a compound of Formula (4). 
Cyclization of compound (4) in the presence of a suitable Lewis acid such as titanium tetracholoride or aluminum chloride or alternatively when Z1 is 3-OR (meta)(where R is C1-5alkyl, or benzyl), trifluoroacetic acid, provides an indanone of the Formula (5). 
Dehydrogenation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in an appropriate solvent or alternatively bromination with pyridinium hydrobromide perbromide in dichloromethane followed by treatment with 1,5-diazabicyclo[4,3,0]non-5-ene provides indenones of Formula (6). 
b) Alternatively, a compound of Formula 6 wherein Z1, Z2 and Z3 are hydrogen and 
can be prepared by treatment of 2-bromobenzoic acid with two equivalents of n-butyllithium in a solvent such as tetrahydrofuran under argon at xe2x88x9278xc2x0 C. followed by the addition of an acid chloride of Formula (7): 
provides a compound of Formula (8): 
Treatment of compounds of type (8) with thionyl chloride at reflux gives an acid chloride which can be isolated by concentration under reduced pressure. This acid chloride can then be treated with diethyl magnesium malonate in a solvent such as ether to give a compound of Formula (9): 
Reaction of a compound of type (9) at reflux with 5% aqueous sodium carbonate gives compounds of Formula (10): 
c) Treatment of an indenone of Formula (11): 
wherein Z1, Z2, Z3 and R1 are as defined for formula I or a group convertable to them, with an organomagnesium compound of Formula (12) wherein R2 is defined for
R2(CH2)nMgBrxe2x80x83xe2x80x83(12)
Formula I or a group convertable to it, in a suitable solvent provides compounds of Formula (13): 
Saponification of compounds of Formula (13) using sodium hydroxide in aqueous methanol followed by reduction with triethylsilane and boron trifluoride etherate in a suitable solvent such as dichloromethane at 0xc2x0 C. affords racemic compounds of Formula (14). 
Conjugate addition of nucleophiles to an ester derived from Formula (14), followed by saponification affords compounds of Formula (I) having an R10 other than hydrogen. Re-introduction of a double bond into an ester derived from such acids followed by conjugate addition of another nucleophilic species and subsequent saponification affords compounds of Formula (1) in which neither R10 substituent is hydrogen.
Reduction of compounds of Formula (13) with triethylsilane and boron trifluoride etherate in a suitable solvent such as dichloromethane at 0xc2x0 C. followed by hydrogenation with hydrogen gas under pressure at approximately 60 psi in the presence of a suitable catalyst such as 10% palladium on charcoal affords compounds of Formula (15): 
Alkylation or acylation of the ester enolate derived from Formula (15) affords compounds wherein P1 and P2 are as defined in Formula (I).
Alternatively, hydrogenation of compounds of Formula (13) with hydrogen gas under pressure at approximately 60 psi in the presence of a suitable catalyst such as 10% palladium on charcoal in a suitable solvent such as ethyl acetate or methanol containing 1-5% acetic acid affords compounds of Formula (15). Treatment of these compounds with a base such as sodium hydroxide in a suitable solvent such as aqueous ethanol provides racemic compounds of Formula (16): 
wherein Z1, Z2 and Z3 are hydrogen; R1=R2; and n is 0. Treatment of compounds of Formula (13) with triethylsilane and boron trifluoride etherate in a suitable solvent such as dichloromethane at 0xc2x0 C. followed by reaction with samarium II iodide in a suitable solvent such as tetrahydrofuran and then saponification, provides compounds of Formula (17) 
With appropriate manipulation and protection of any chemical functionalities, synthesis of the remaining compounds of the Formula (I) is accomplished by methods analogous to those above and to those described in the Experimental section.
In order to use a compound of the Formula (I) or a pharmaceutically acceptable salt thereof for the treatment of humans and other mammals it is normally formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition.
Compounds of Formula (1) and their pharmaceutically acceptable salts may be administered in a standard manner for the treatment of the indicated diseases, for example orally, parenterally, sub-lingually, transdermally, rectally, via inhalation or via buccal administration.
Compounds of Formula (1) and their pharmaceutically acceptable salts which are active when given orally can be formulated as syrups, tablets, capsules and lozenges. A syrup formulation will generally consist of a suspension or solution of the compound or salt in a liquid carrier for example, ethanol, peanut oil, olive oil, glycerine or water with a flavouring or colouring agent. Where the composition is in the form of a tablet, any pharmaceutical carrier routinely used for preparing solid formulations may be used. Examples of such carriers include magnesium stearate, terra alba, talc, gelatin, agar, pectin, acacia, stearic acid, starch, lactose and sucrose. Where the composition is in the form of a capsule, any routine encapsulation is suitable, for example using the aforementioned carriers in a hard gelatin capsule shell. Where the composition is in the form of a soft gelatin shell capsule any pharmaceutical carrier routinely used for preparing dispersions or suspensions may be considered, for example aqueous gums, celluloses, silicates or oils and are incorporated in a soft gelatin capsule shell.
Typical parenteral compositions consist of a solution or suspension of the compound or salt in a sterile aqueous or non-aqueous carrier optionally containing a parenterally acceptable oil, for example polyethylene glycol, polyvinylpyrrolidone, lecithin, arachis oil, or sesame oil.
Typical compositions for inhalation are in the form of a solution, suspension or emulsion that may be administered as a dry powder or in the form of an aerosol using a conventional propellant such as dichlorodifluoromethane or trichlorofluoromethane.
A typical suppository formulation comprises a compound of Formula (1) or a pharmaceutically acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent, for example polymeric glycols, gelatins, cocoa-butter or other low melting vegetable waxes or fats or their synthetic analogues.
Typical transdermal formulations comprise a conventional aqueous or non-aqueous vehicle, for example a cream, ointment, lotion or paste or are in the form of a medicated plaster, patch or membrane.
Preferably the composition is in unit dosage form, for example a tablet, capsule or metered aerosol dose, so that the patient may administer to themselves a single dose.
Each dosage unit for oral administration contains suitably from 0.1 mg to 500 mg/Kg, and preferably from 1 mg to 100 mg/Kg, and each dosage unit for parenteral administration contains suitably from 0.1 mg to 100 mg/Kg, of a compound of Formula (1) or a pharmaceutically acceptable salt thereof calculated as the free acid. Each dosage unit for intranasal administration contains suitably 1-400 mg and preferably 10 to 200 mg per person. A topical formulation contains suitably 0.01 to 5.0% of a compound of Formula (I).
The daily dosage regimen for oral administration is suitably about 0.01 mg/Kg to 40 mg/Kg, of a compound of Formula (I) or a pharmaceutically acceptable salt thereof calculated as the free acid. The daily dosage regimen for parenteral administration is suitably about 0.001 mg/Kg to 40 mg/Kg, of a compound of the Formula (I) or a pharmaceutically acceptable salt thereof calculated as the free acid. The daily dosage regimen for intranasal administration and oral inhalation is suitably about 10 to about 500 mg/person. The active ingredient may be administered from 1 to 6 times a day, sufficient to exhibit the desired activity.
No unacceptable toxicological effects are expected when compounds of the invention are administered in accordance with the present invention.
The biological activity of the compounds of Formula (I) are demonstrated by the following tests:
I. Binding Assay
A) Membrane Preparation (Rat cerebellum or kidney cortex)
Rat cerebellum or kidney cortex were rapidly dissected and frozen immediately in liquid nitrogen or used fresh. The tissues, 1-2 g for cerebellum or 3-5 g for kidney cortex, were homogenized in 15 mls of buffer containing 20 mM Tris HCl and 5 mM EDTA, pH 7.5 at 4xc2x0 C. using a motor-driven homogenizer. The homogenates were filtered through cheesecloth and centrifuged at 20,000xc3x97g for 10 minutes at 4xc2x0 C. The supernatant was removed and centrifuged at 40,000xc3x97g for 30 minutes at 4xc2x0 C. The resulting pellet was resuspended in a small volume of buffer containing 50 mM Tris, 10 mM MgCl2, pH 7.5; aliquotted with small vials and frozen in liquid nitrogen. The membranes were diluted to give 1 and 5 micrograms of protein for each tube for cerebellum and kidney cortex in the binding assay.
Freshly isolated rat mesenteric artery and collateral vascular bed were washed in ice cold saline (on ice) and lymph nodes were removed from along the major vessel. Then, the tissue was homogenized using a polytron in buffer containing 20 mM Tris and 5mM EDTA, pH 7.5 at 4xc2x0 C. in 15 ml volume for xcx9c6 gm of mesenteric artery bed. The homogenate was strained through cheesecloth and centrifuged at 2,000xc3x97g for 10 min. at 4xc2x0 C. The supernatant was removed and centrifuged at 40,000xc3x97g for 30 min. at 4xc2x0 C. The resulting pellet was resuspended as explained above for cerebellum and kidney cortex. Approximately 10 micrograms of membrane protein was used for each tube in binding experiments.
B) CHO Cell Membrane Preparation
CHO cells stably transfected with human ETA and ETB receptors were grown in 245 mmxc3x97245 mm tissue culture plates in Dulbecco""s modified Eagle""s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The confluent cells were washed with DPBS (Dulbecco""s phosphate buffered saline) containing protease inhibitor cockatil (5 mM EDTA, 0.5 mM PMSF, 5 ug/ml leupeptin, and 0.1 U/ml aprotinin) and scraped in the same buffer. After centrifugation at 800xc3x97g, the cells were lysed by freezing in liquid nitrogen and thawing on ice followed by homogenization (30 times using glass dounce homogenizer) in lysis buffer containing 20 mM Tris HCl, pH 7.5 and the protease inhibitor cocktail. After an initial centrifugation at 800xc3x97g for 10 min to remove unbroken cells and nuclei, the supernatants were centrifuged at 40,000xc3x97g for 15 min and the pellet was resuspended in 50 mM Tris HCl, pH 7.5 and 10 mM MgCl2 and stored in small aliquots at xe2x88x9270xc2x0 C. after freezing in liquid N2. Protein was determined using BCA method and bovine serum albumin as the standard.
C) [125I]ET-1 Binding Protocol
[125I]ET-1 binding to membranes from rat cerebellum (2-5 mg protein/assay tube) or kidney cortex (3-8 micrograms protein/assay tube) or CHO cell membranes (containing 4-6 and 1-2 micrograms of membrane protein for ETA and ETB receptors, respectively) were measured after 60 minutes incubation at 30xc2x0 C. in 50 mM Tris HCl, 10 mM MgCl2, 0.05% BSA, pH 7.5 buffer in a total volume of 100 microliters. Membrane p rotein was added to tubes containing either buffer or indicated concentration of compounds. [125I]ET-1 (2200 Ci/mmol) was diluted in the same buffer containing BSA to give a final concentration of 0.2-0.5 nM ET-1. Total and nonspecific binding were measured in the absence and presence of 100 nM unlabelled ET-1. After the incubation, the reactions were stopped with 3.0 ml cold buffer containing 50 mM Tris and 10 mM MgCl2, pH 7.5. Membrane bound radioactivity was separated from free ligand by filtering through Whatman GF/C filter paper and washing the filters 5 times with 3 ml of cold buffer using a Brandel cell harvester. Filter papers were counted in a gamma counter with an efficiency of 75%. IC50""s for the compounds of this invention range from 0.01 nm to 50 uM.
II. In Vitro Vascular Smooth Muscle Activity
Rat aorta are cleaned of connective tissue and adherent fat, and cut into ring segments approximately 3 to 4 mm in length. Vascular rings are suspended in organ bath chambers (10 ml) containing Krebs-bicarbonate solution of the following composition (millimolar): NaCl, 112.0; KC1, 4.7; KH2PO4, 1.2; MgSO4, 1.2; CaCl2, 2.5; NaHCO3, 25.0; and dextrose, 11.0. Tissue bath solutions are maintained at 37xc2x0 C. and aerated continuously with 95% O2/5% CO2. Resting tensions of aorta are maintained at 1 g and allowed to equilibrate for 2 hrs., during which time the bathing solution is changed every 15 to 20 min. Isometric tensions are recorded on Beckman R-611 dynographs with Grass FT03 force-displacement transducer. Cumulative concentration-response curves to ET-1 or other contractile agonists are constructed by the method of step-wise addition of the agonist. ET-1 concentrations are increased only after the previous concentration produces a steady-state contractile response. Only one concentration-response curve to ET-1 is generated in each tissue. ET receptor antagonists are added to paired tissues 30 min prior to the initiation of the concentration-response to contractile agonists.
ET-1 induced vascular contractions are expressed as a percentage of the response elicited by 60 mM KCl for each individual tissue which is determined at the beginning of each experiment. Data are expressed as the mean xc2x1S.E.M. Dissociation constants (Kb) of competitive antagonists were determined by the standard method of Arunlakshana and Schild. The potency range for compounds of this invention range from 0.01 nM to 50 uM.